The long term objective of this project is to develop a vaccine to protect humans from antigenically different forms of HIV-1 found in nature. Such protection will require elicitation of immune responses against diverse HIV- 1 envelop proteins. We submit that multiple HIV-1 envelope proteins must be presented in a vaccine to induce immunological memory against the range of HIV-1 variants. We further proposed that the magnitude of diverse responses may be optimized by utilization of more than one vaccine delivery system. Our pre-clinical studies evaluation a three-tired vaccine delivery system with (i) recombinant DNA vaccine followed by (ii) recombinant vaccinia virus vaccine followed by (iii) purified protein vaccine (D-V-P) demonstrate high levels of HIV-1 neutralizing antibodies in mice. The goal of this project is to translate this vaccine strategy to human application. A multi-envelope recombinant vaccinia virus vaccine (PolyEnv1) is currently in clinical trial (Project 4). Accordingly, in this project we will prepare DNA and purified proteins fro use in humans. The first two aims of this project describe the design and production of clinical grade material. While technically driven these two aims are critical for the conduct of small scale human trials (Project 4). The third aim tests the hypothesis that these aims are critical for the conduct of small scale human trials (Project). The third aim tests the hypothesis that our 3-tiered, multi- envelope vaccine strategy can protect non-human primates from heterologous virus. The implementation of this aim is critical for clinical advanced of the vaccine approach. Specific aims developed in this project are: Aim 1: To prepare a multi-envelope DNA vaccine that is safe, immunogenic and suitable for use in humans. Aim 2: To prepare a multi-envelope protein vaccine that is safe, immunogenic and suitable for use in humans. Aim 3: To determine whether a three-tiered multi-envelope vaccine protects non-human primates from heterologous virus challenge.